It is the objective of the work proposed in this grant application to enhance our understanding of the mechanisms by which poliovirus (PV), an enterovirus belonging to the Picornaviridae, encapsidates its genome. This virus family includes a large number of human and animal pathogens that cause more than 6 billion human infections worldwide each year. These infections lead to a variety of diseases ranging from the mild (common cold) to the serious (poliomyelitis). In spite of research for many years the details of most steps in the life cycle of PV remain unknown. However, the proliferation of PV remains an important medical issue because epidemic PV infections are expected to occur even after the circulation of wt PV is interrupted globally. This proposal can be divided into 3 parts. The first aim deals with the development of new genetic tools to study the genetics of encapsidation of chimeras constructed from PV and the closely related C-cluster coxsackie A viruses. We expect that the morphogenesis phenotypes of the chimeric viruses will be very useful in analyzing the interaction of capsid and nonstructural proteins during encapsidation. The second aim of the proposal deals with two specific inhibitors of enterovirus encapsidation, hydantoin and L-buthionine-sulfoximine (BSO), whose effect is expected to block encapsidation at different stages. In this study PV, CAVs and PV/C-CAV chimeras will be used to identify and analyze escape mutants in proteins involved in encapsidation. In our third aim we propose to analyze the role in morphogenesis of non-structural proteins 2CATPase, 3CDpro, VPg, and, and search for an elusive RNA encapsidation signal. We plan to use both genetic and biochemical studies to analyze the role of nonstructural proteins 2CATPase and 3CDpro in encapsidation. The role of VPg will be tested by a large-scale scan of hundreds of VPg mutants with the aim of finding replication positive but encapsidation negative mutants. Finally, we will use a novel strategy (codon-pair optimization) to scan the PV RNA for an elusive encapsidation signal. Since encapsidation is a uniquely viral process an understanding of its mechanism will aid the development of antiviral drugs that target this particular step in the enteroviral life cycle. The 2CATPase and 3CDpro proteins are highly conserved among enteroviruses hence they provide an excellent target for drug development to treat multiple enteroviral diseases. It is believed that these studies and results will be of interest not only to those investigators who study the encapsidation of other enteroviruses but also to those who are interested in picornaviruses or RNA viruses in general.